The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome

The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation and activity towards transcription or replication of the viral genome, respectively. Here, we provide evidence that the FluPol-RNAP II binding interface, beyond its well-acknowledged function in cap-snatching during transcription initiation, has also a pivotal role in replication of the viral genome. Using a combination of cell-based and in vitro approaches, we show that the RNAP II C-terminal-domain, jointly with ANP32, enhances FluPol replication activity. We observe successive conformational changes to switch from a transcriptase to a replicase conformation in the presence of the bound RNPAII C-terminal domain and propose a model in which the host RNAP II is the anchor for transcription and replication of the viral genome. Our data open new perspectives on the spatial coupling of viral transcription and replication and the coordinated balance between these two activities.

6RR7, [https://www.rcsb.org/structure/6rr7] 1 ), with PA (green), PB1 (grey), and PB2 (blue).Key FluPol CTDbinding residues are highlighted in red.(B-C) Lysates of HEK-293T cells transiently expressing for WSN PB1, PB2 and PA with the indicated mutation were analysed by western blot using the indicated antibodies (n=1).(D) Cell-based WSN FluPol binding and activity assays as in Fig. 1B.Left Y-axis (linear scale): FluPol binding to the CTD, huANP32A and chANP32A.Right Y-axis (logarithmic scale): FluPol activity (hatched bars).Luminescence signals are represented as a percentage of PA WT (mean ± SD, n=3, 4, 3, 4, *p < 0.033, **p < 0.002, ***p < 0.001, one-way ANOVA; Dunnett's multiple comparisons test).(E) WSN vRNPs with the indicated PA mutations were reconstituted in HEK-293T cells using the NA vRNA segment.Steady-state levels of NA mRNA, cRNA and vRNA were quantified by strand-specific RT-qPCR 2 and are presented as ratios of mRNA to vRNA (grey bars) or cRNA to vRNA (blue bars) levels relative to PA WT (mean ± SD, n=3, ***p < 0.001, one-way ANOVA; Dunnett's multiple comparisons test).RNA levels are presented in Fig. 1C.(F) Cell-based Anhui-H7N9 FluPol binding and activity assays using a plasmid encoding PA in which the PA-X (PA-DX) ORF frame was deleted as described previously 3 , as in   (F) For each initial FluPol mutant and representative FluPol genotype observed during serial passaging, FluPol binding to RED (mean ± SD, n=4, x-axis) was plotted against FluPol activity (mean ± SD, n=4, y-axis).Combinations of mutations which appeared during passaging of the WSN PA K289A, R454A, K635A and R638A mutant viruses are highlighted in grey, green, blue and pink respectively.r: Pearson correlation coefficient (twotailed 95% confidence interval).Source data are provided as a Source data file.FluPol second-site mutations were displayed as spheres in orange on the FluPol(R) and in yellow on the FluPol(E), respectively, and are labelled in grey (PB1), blue (PB2) or green (PA).(C) Representation of the Cryo-EM structure of the FluPol heterotrimer in the FluPol(T) conformation, bound to a 3'5' vRNA promoter and a short capped RNA primer (A/NT/60/1968, PDB: 6RR7 [https://www.rcsb.org/structure/6rr7], 1 ).Ribbon diagram representation with PA in green, PB1 in grey, and PB2 in blue.Initially mutated FluPol residues (PA K289A, R454A, K635A and R638A) are displayed as spheres in red and residues corresponding to FluPol second-site mutations in pink.(D) Residues corresponding to NP second-site mutations were mapped on the RNA-bound crystal structure of a monomeric NP mutant (PDB: 7DXP, [https://www.rcsb.org/structure/7DXP] 6).Ribbon diagram representation with NP in salmon, RNA in yellow and residues corresponding to NP second-site mutations are displayed as spheres in orange.(D) NGS sample preparation of FluPol Zheijiang-H7N9 4M de novo replication products.Urea-PAGE gel stained with SYBR-Gold of the equivalent de novo reaction (Fig. S6B lane 8) before (-) and after (+) 5'-monophosphate (5'-p) RNA digestion using a terminator 5'-p-dependent exonuclease.The dotted rectangle corresponds to the sample sent for NGS.The decade molecular weight marker (Nts) is shown on the left side of the gel.(E) RNA-sequencing analysis of FluPol Zheijiang-H7N9 4M de novo replication products in the presence of pS5 CTD(6mer) and huANP32A.After trimming, the number of reads is plotted according to their lengths.Source data are provided as a Source data file.(Right) Coulomb potential of the v51_mut_S isolated from the cryo-EM map of FluPol Zhejiang-H7N9 4M in pre-initiation state (mode A) without PB2-C, at an overall resolution of 2.5 Å. 3'-U1 is displayed but not visible in the cryo-EM map.3'-G3 is in the +1 active site position.(B) (Left) Schematics of the v51_mut_S template and the de novo cRNA replication product (33-mer) conformations present in the cryo-EM map of FluPol Zhejiang-H7N9-4M stalled in an elongation state using UpNHpp.The template is coloured as in A. The de novo cRNA product is coloured in blue.Unseen nucleotides are in italics.The +1 active site position is indicated.(Right) Coulomb potential of the v51_mut_S template and the de novo cRNA replication product (33-mer, 32-mer if A1 is not incorporated) isolated from the cryo-EM map of FluPol Zhejiang-H7N9-4M stalled in an elongation state using UpNHpp, at an overall resolution of 2.9 Å.The non-hydrolysable UpNHpp is in the +1 active site position, coloured in dark grey and circled by a dotted line.Flexible nucleotides are shown as dotted lines.

Figure S3 :
Figure S3: Serial passaging of mutant viruses with mutations at the FluPol-CTD interface selects for adaptive mutations which restore FluPol binding to the CTD and huANP32A (A) Plaque phenotypes of passaged WSN mutant viruses.Representative images of crystal violet-stained plaque assays on reverse genetics (RG), p1, p4 and p8 supernatants are shown.(B) Short-read next generation sequencing of the viral genome.Second site mutations found in !10% of reads in at least one passage are shown (green circles).The fraction of reads showing a given mutation is indicated by

Figure S4 :
Figure S4: Representation of FluPol and NP second-site mutations which were selected during serial cell culture passaging of PA mutant viruses (A) Second-site mutations which were selected on FluPol and NP during serial passaging of recombinant plaquepurified PA mutant (K289A, R454A, K635A, R638A) viruses were mapped on a linear domain representation of PB2, PB1, PA and NP.Only second-site mutations found in ≥ 10% of reads in at least one passage are shown.(B) Model of the FluAPol replication complex (FluPol(R), FluPol(E) and ANP32A) based on a FluCPol Cryo-EM structure (C/Johannesburg/1/1966 structure, PDB: 6XZR, [https://www.rcsb.org/structure/6XZR] 5 ).Ribbon diagram representation with PA in green, PB1 in grey, PB2 in blue and ANP32A in pink.Initially mutated FluPol residues (PA K289A, R454A, K635A and R638A) are displayed as spheres in red.Residues corresponding to

Figure S6 :
Figure S6: FluPol replication activity is enhanced in the presence of CTD and huANP32A (A-B) De novo replication activity of (A) Zhejiang-H7N9 WT and (B) Zhejiang-H7N9 4M (PA E349K, PA R490I, PB1 K577G, and PB2 G74R) using the v51_mut_S template in the presence of either 3 NTPs (AUG) or 4 NTPs (AUGC), with or without pS5 CTD(6mer) and huANP32A FL (left) or different huANP32A truncation mutants (right) as defined in Fig. 3E.Tentative full-length and stalled replication products are indicated by an

Figure S7 :
Figure S7: Cryo-EM image processing flowchart to obtain FluPol Zhejiang-H7N9-4M de novo replication structures (sample 1| Krios dataset) Schematics of the image processing strategy used to obtain (i) the Cryo-EM map of FluPol Zhejiang-H7N9-4M in a pre-initiation state (mode A), with PB2-C in a replicase conformation, (ii) the cryo-EM map of FluPol Zhejiang-H7N9-4M in pre-initiation state (mode A) without PB2-C, and (iii) the cryo-EM map of FluPol Zhejiang-H7N9-4M stalled in an elongation state using UpNHpp.Further processing steps for the dislocated FluPols are not shown.Representative cropped micrograph from the TEM Titan Krios dataset, 2D class averages, 3D class averages and intermediate structures are displayed.CTF stands for "Contrast Transfer Function".The final number of particles, filtered EM maps according to the local resolution and corresponding Fourier Shell Correlation curves (FSC) are shown.Scale bar = 200 Å.

Figure S8 :
Figure S8: RNA conformation schematics, densities and models of the different states obtained (A) (Left) Schematics of the v51_mut_S template conformation present in both (i) the cryo-EM map of FluPol Zhejiang-H7N9-4M in pre-initiation state (mode A) without PB2-C or (ii) with PB2-C in replicase conformation.The 5' vRNA end (1-30) is coloured in pink.The 3' vRNA end (31-51) is coloured in gold.Introduced mutations are coloured in red.Unseen nucleotides are in italics.The +1 active site position is indicated by a dotted rectangle.(Right)Coulomb potential of the v51_mut_S isolated from the cryo-EM map of FluPol Zhejiang-H7N9 4M in pre-initiation state (mode A) without PB2-C, at an overall resolution of 2.5 Å. 3'-U1 is displayed but not visible in the cryo-EM map.3'-G3 is in the +1 active site position.(B) (Left) Schematics of the v51_mut_S template and the de novo cRNA replication product (33-mer) conformations present in the cryo-EM map of FluPol Zhejiang-H7N9-4M stalled in an elongation state using UpNHpp.The template is coloured as in A. The de novo cRNA product is coloured in blue.Unseen nucleotides are in italics.The +1 active site position is indicated.(Right) Coulomb potential of the v51_mut_S template and the de novo cRNA replication product (33-mer, 32-mer if A1 is not incorporated) isolated from the cryo-EM map of FluPol Zhejiang-H7N9-4M stalled in an elongation state using UpNHpp, at an overall resolution of 2.9 Å.The non-hydrolysable UpNHpp is in the +1 active site position, coloured in dark grey and circled by a dotted line.Flexible nucleotides are shown as dotted lines.

Figure S9 :
Figure S9: Cryo-EM image processing flowchart to obtain FluPol Zhejiang-H7N9-4M self-stalled elongation structure (sample 2 | Glacios) (A) Schematics of the image processing strategy used to obtain the cryo-EM map of FluPol Zhejiang-H7N9 in self-stalled elongation state.Representative cropped micrograph from the TEM Glacios dataset, 2D class averages, 3D class averages are displayed.CTF stands for "Contrast Transfer Function".The final number of particles, filtered EM maps according to the local resolution and corresponding Fourier Shell Correlation curves (FSC) are shown.Scale bar = 200 Å.(B) Schematics of the v51_mut_S template and the de novo cRNA replication product (35-mer or 34-mer if A1 is not incorporated) conformations present in the cryo-EM map of FluPol Zhejiang-H7N9-4M self-stalled in

Figure S10 :
Figure S10: Cryo-EM image processing flowchart to obtain FluPol Zhejiang-H7N9 PA K289A+C489R structure (sample 3 | Glacios) Schematics of the image processing strategy used to obtain the cryo-EM map of FluPol Zhejiang-H7N9 PA K289A+C489R.Representative cropped micrograph from the TEM Glacios dataset, 2D class averages, 3D class averages and intermediate structures are displayed.CTF stands for "Contrast Transfer Function".The final number of particles, filtered EM maps according to the local resolution and corresponding Fourier Shell Correlation curves (FSC) are shown.Scale bar = 200 Å.

Figure S11 :
Figure S11: Restoration of CTD-binding Site 2A rescues FluPol replication activity and enhances FluPol binding to huANP32A (A) Recombinant WSN mutant viruses were produced by reverse genetics.Representative plaque assays as quantified in Fig. 5D of mutant viruses harbouring PA K289A and the second-site mutation PA C489R are shown.The supernatants of RG experiments (n=2) were titrated on MDCK cells and stained by crystal violet.(B) WSN vRNPs with the PA K289A mutation or the double mutation PA K289A+C489R were reconstituted in HEK-293T cells by transient transfection using a plasmid encoding the NA vRNA segment.Steady-state levels of NA mRNA, cRNA and vRNA were quantified by strand-specific RT-qPCR 2 normalised to GAPDH by the 2 −ΔΔCT method 8 and are represented as a percentage of PA WT. (mean ± SD, n=3, *p < 0.033, **p < 0.002, ***p < 0.001, one-way ANOVA; Dunnett's multiple comparisons test).(C)HEK-293T cells were co-transfected with expression plasmids for WSN PB1, PB2 and PA with the indicated mutation.Cell lysates were analysed by western blot using antibodies specific for PA and tubulin (n=1).(D) Anhui-H7N9 FluPol with the PA K289A mutation and the second-site mutation PA C489R were characterised.(left): Anhui-H7N9 FluPol activity was measured by vRNP reconstitution (PB2, PB1, PA-DX, NP) in HEK-293T cells, using a model vRNA encoding the Firefly luciferase.Luminescence was measured and normalised to a transfection control.The data are represented as a percentage of PA WT. (middle): Anhui-H7N9 FluPol binding to the CTD was assessed using a split-luciferase-based complementation assay.HEK-293T cells were co-transfected with expression plasmids for the CTD tagged with one fragment of the G. princeps luciferase and FluPol tagged with the other fragment.Luminescence due to luciferase reconstitution was measured and the data are represented as a percentage of PA WT. (right): huANP32A was tagged with one fragment of the G. princeps luciferase and binding to Anhui-H7N9 FluPol was determined as for FluPol binding to the CTD.(mean ± SD, n=4, 5, 4, *p < 0.033, ***p < 0.001, one-way ANOVA; Dunnett's multiple comparisons test).Source data are provided as a Source data file.

Figure S12 :
Figure S12: Restoration of CTD-binding Site 1 rescues FluPol replication activity and enhances FluPol binding to huANP32A (A) (left) Cartoon representation of the pS5 CTD bound to FluPol Zhejiang-H7N9 4M (replicase-like conformation) in CTD-binding site 1A.Green: PA subunit; Red: pS5 CTD; yellow dashed lines : putative hydrogen bonds between PA R638 and pS5 (corresponding distances are indicated).The Coulomb potential map of PA C453, PA R638, and pS5 is shown.(right) Model derived from FluPol Zhejiang-H7N9 4M structure bearing PA C453R and PA R638A mutations.Green: PA subunit; red: pS5 CTD.PA C453R and PA R638A residues are displayed.Hypothetical distances between PA C453R and pS5 are shown.(B-F) Phenotypes associated with the WSN FluPol PA R638A mutation and the second-site mutation PA C453R.(B-C) Plaque phenotype of recombinant WSN mutant viruses produced by reverse genetics (n=2).RG supernatants were titrated on MDCK cells and stained by crystal violet.Plaque diameters (mm) were measured and each dot represents one viral plaque.Representative plaque assays are shown in (C) (#) pinhead-sized plaques.(D) WSN FluPol activity was measured by vRNP reconstitution in HEK-293T cells using a model Fluc-vRNA.Luminescence signals are represented as a percentage of PA WT (mean ± SD, n=3).(E) HEK-293T cells were co-transfected with expression plasmids for WSN PB1, PB2 and PA with the indicated mutation.Cell lysates were analysed by western blot using the indicated antibodies (n=1).(F) WSN FluPol binding to the huANP32A was assessed using a split-luciferase-based complementation assay.Luminescence signals are represented as a percentage of PA WT. (mean ± SD, n=4, *p < 0.033, **p < 0.002, one-way ANOVA; Dunnett's multiple comparisons test).Source data are provided as a Source data file.